In this study we evaluated the use of nested PCR utilizing the ITS1 and ITS2 regions as targets for diagnosis and direct differentiation of Leishmania donovani. AlsoPCR products of gp63 and anonymous regions were attempted for the characterization and identification of intraspecific polymorphisms of the causative agent of visceral leishmaniasis (VL) in eastern Sudan from clinical samples spotted on filter papers using SSCP technique. Also we assessed DNA polymorphisms among 23 cultured L. donovani isolates from eastern Sudan, one from Kenya, one from India and one from China with 4 different PCR based approaches as tools to detect polymorphisms.

The present study showed that PCR of both inguinal lymph node and bone marrow aspirates is significantly more positive than microscopy (tables 4 & 5). No difference was detected between microscopy and PCR when the clinical materials were taken from patients with confirmed cases of VL.This is in agreement with Osman et al. (1997). However, Leishmania DNA was demonstrated in 48.3% of VL suspect cases, thereby confirming the clinical diagnosis. This indicates that the main advantage of using PCR as a diagnostic tool is for VL suspects.

The sensitivity of microscopic examination of smears from lymph node varies from 78% (Siddig et al., 1989) to 58.3% (Zijlstra et al., 1992), while the sensitivities of microscopy of splenic and bone marrow aspirates were 96.4 and 70.2% (Zijlstra et al., 1992). This is congruent with the findings of this study, where 30% of the clinical samples from lymph node aspirates that were found to be negative on basis of microscopy, were positive when the aspirates were taken from bone marrow. This observation indicates that the significance of microscopy results should be judged carefully against the types of the clinical material.

When 3 clinical samples were taken from lymph node, bone marrow and skin from post kala-azar dermal leishmaniasis (PKDL) patient and PCR was performed for these samples, a positive result was obtained only from the skin sample. Although one sample is not enough to draw a conclusion, the same result was obtained by Osman etal. (1998) who found that only 2 of 18 bone marrow aspirates from PKDL patients to be positive in PCR. This is probably due to the clearance of parasites from the lymph node and bone marrow as cell-mediated immunity develops during or after treatment of VL.

PCR of the entire ITS region of L. donovani was found to be less sensitive than PCR of the 18S ribosomal RNA described by Osman et al., (1997 & 1998). In spite of this, ITS region is targeted in a nested PCR (ITS1 & ITS2) because sequences of this region are more variable and allow clear species identification by RFLP or SSCP and also strain differences could be expected.

Various DNA techniques have been valuable for the study of genetic variation, and their application has provided a wealth of molecular data (McManus & Bowles, 1996). Intraspecific variation in SSCP banding patterns was clearly observed in the ITS1 region with 12 profiles detected among Sudan isolates, with profile A being dominant (76.7%), and 2 ITS1 –SSCP profiles observed among the samples from Kenya, India and China. On the other hand, all but one of the Sudanese samples had the same ITS2 – SSCP pattern (A). Isolates of the same species (L. donovani) but of different geographical origin had showed varying ITS2-SSCP patterns. We may conclude that ITS2-SSCP pattern analysis could be a useful method for differentiation of L. donovani strains from different geographical areas. To consolidate this observation, more samples from different geographical origins are required.

Iwahana et al. (1992) claim that PCR-SSCP analysis is only effective for DNA fragments of less than 300 to 400 bp. Therefore the ITS2 region was further divided into 2 regions (ITS 2A and ITS 2B), using internal primers. Again the same above result was obtained. This means that ITS2 sequence seems to be highly conserved in all investigated L. donovani isolates from Sudan, with the exception of only one sample. In contrast, ITS1 region appears to undergo more rapid evolutionary changes and may vary among populations of the same species of the same geographical origin. The polymorphisms in the ITS1 observed in this study were independent of the clinical symptoms, of the stage of the disease and of the tribe of the patient.

DNA fragments showing different SSCP patterns were further characterized by radioactive cycle sequencing to determine the underlying nucleotide polymorphisms. Different patterns in the ITS1 region were mainly due to deletion of Adenine residues from A stretches or AT pairs (Fig.11). This may be attributed to the weak AT double bond. Patterns L, H and E have a unique part in their sequences where different nucleotides were located at the same position. This is already clearly indicated in the SSCP analysis where patterns L and H consist of multiple bands (Fig.8a). This may be attributed to the fact that the ITS region is a multi-copy gene and heterogeneity may also arise between individual copies of the ribosomal operon, but mixed infections or hybrids by different strains of L. donovani can not be excluded. The results obtained by SSCP for ITS2 were also confirmed by DNA sequencing.

Intraspecies variations in SSCP banding patterns were clearly observed in the coding region of gp63 with 3 profiles detected among 31 Sudanese L. donovani isolates. These results were also confirmed by DNA sequencing. The gp63 molecule participates in the parasites initial attachment to mammalian macrophages and likely contributes to their survival within the macrophage phagolysosomes (Bordier, 1987; Chang et al., 1990). This finding indicates that gp63 could be a potential useful marker for study of polymorphisms. Those polymorphisms can possibly correlated to the severity of the disease The same result was obtained by Victoir et al. (1998) who illustrates the very high genomic and genetic plasticity of gp63 genes revealed by gp63-RFLP and PCR-RFLP in 4 species of subgenus viannia and the possibility to relate these genetic differences to phenotypic properties, such as pathogenicity.

Six amplified anonymous regions of DNA were screened for polymorphisms with the SSCP technique from isolates spotted on filter papers. Unfortunately good SSCP results were obtained from only one region. In contrast to this finding, good PCR products and SSCP polymorphic patterns were observed among cultured Sudanese L. donovani (DON12- DON23) using the same anonymous primers (Lewin, 2000). This difference may be due to the fact that, these anonymous regions represent single copy genes and hence the SSCP was hampered by the little amount of PCR products obtained from samples spotted on filter papers. At the same time good results were obtained when SSCP was performed for 8 of the same above isolates but from cultured parasites. We may conclude that anonymous regions could be good for population genetics studies taking into consideration, they are single copy genes and thus, can address for genetic diploidy and sexual reproduction of Leishmania provided that there is large amount of PCR products.

No intra-specific variation was observed in the RFLP patterns when the amplified ITS regions of different strains of L donovani, were digested with a panel of different frequently cutting restriction enzymes (Fig. 7). This may be explained by the fact that sequence variation may go undetected by RFLP analysis since the restriction enzymes survey only a subset of the total variable sites (Stothard et al., 1997).

In contrast to this observation, considerable heterogeneity was found within the ITS-RFLP of strains of L. tropica and L. aethiopica (Schönian et al., 2000 & 2001) and of New World species of Leishmania whereas different species showed different levels of variation (Guevara et al. 1992; Cupolillo, et al. 1995). These findings reveal that different levels of heterogeneity occur within different species of Leishmania.

When PCR-fingerprinting approach was done on cultured parasites, highly similar PCR profiles were observed between all tested isolates (Figs. 19&20); thus confirming previous results (Schönian et al., 1996; Oskam et al., 1998; Lewin, 2000). This may be attributed to the fact that the fingerprinting technique is less discriminatory because mutations can be detected only if they affect primer-binding sites or if they lead to bigger insertions/deletions that would change the fragment size. Gasser (1997) also reported that fragments of the same size but differing in sequence will co-migrate within one band in a gel, and may be misinterpreted as the same sequence.

We can conclude that SSCP is advantageous relative to both PCR-RFLP and PCR-fingerprinting approaches for the detection of sequence variation in rRNA genes within the L. donovani species. The SSCP technique is sensitive to detect genetic diversity that differed by only one nucleotide (Fig. 12 sequences, LdX & LdY).

In addition, ITS-SSCP is a technique that can be performed easily and rapidly, directly from blotted clinical samples infected with Leishmania parasites without prior cultivation of the parasite, unlike PCR-fingerprinting. Samples collected under field conditions where cultures are vulnerable to contamination can be analysed directly by PCR-SSCP methods in specialized laboratories. Further more, by cultivating the leishmania parasite it is possible that mixed infections will be missed due to different growth rates of different strains in blood agar culture (Ibrahim et al., 1994). Thus SSCP can be an excellent method for the screening of genetic polymorphisms within the genus Leishmania because it can be used with low parasite numbers and contaminated samples. Samples showing different SSCP patterns could be subsequently sequenced. Automated direct sequencing of the PCR product without prior screening by the SSCP technique might be profitable for the detection of polymorphic sequences in single copy genes. However with multicopy genes, SSCP is of advantage because direct sequencing is hampered by possible sequence differences among the individual copies (see patterns E, H, and L in this study).

In this study, of 40 cultures attempts in the field only 8 were successful. This was due to problems with contamination (aseptic techniques in the field were not ideal as in the equipped laboratory), problems with the conservation of adequate media, and problems with maintenance of the optimal temperature under field conditions. Simultaneously, we were able to apply ITS-SSCP for these 8 samples directly from collections obtained by spotting of the lymph node or bone marrow aspirates on filter papers. Although SSCP has been used to detect sequence variation in ITS, gp63 and anonymous regions of L. donovani, this method could be a powerful analytical tool also for other parasitic organisms. SSCP can also be used for the detection of strain-specific polymorphisms not only in the above mentioned targets but also in any already published sequence and thus lessen the dependence on reference laboratories for identification of Leishmania strains.

No correlation was however discerned between the PCR-SSCP polymorphic patterns and the clinical manifestation of the human disease (6PKDL, 83 VL and 1 CL patients). Also Schönian et al., (2000, 2001) found no correlation between different ITS-RFLP, fingerprinting patterns and the clinical manifestation of the disease caused by the L. aethiopica and also the disease caused by L. tropica. It would be interesting to determine whether L. donovani causing mucosal leishmaniasis, PKDL and VL in Sudan could be correlated with specific SSCP patterns, may be other gene (i.e. virulent genes that still have to be identified) could be used as a target in further study.

There was male predominance among the patients in this study area. This may be due to the male greater outdoor exposure to sandfly and the greater value placed on male lives, which bring them in close contact with the forest. The high infection rate among children may indicate that some infections are domestic or peridomestic. Most of the patients in this area were from low socio-economic class, anaemic and this may indicate that leishmaniasis has characteristics of an opportunistic disease too.

Lastly we can emphasise that, it is a high time in Sudan to find a quick accurate tool for differential diagnosis between endemic diseases resembling clinically Leishmaniaasis, like chronic malaria, typhoid, brucellosis, trypanosomiasis. PCR diagnosis evaluated in this study can be used as a supplement to the existing gold standard microscopic method, especially for leishmaniasis suspect cases. This will help in prescribing accurate treatment, improving quality of the care of the patient and reduce the morbidity and mortality rates. Also it can be applied to samples collected from vector or reservoir host to support control program.

The advantage of the PCR products investigated in this study is that can be used for epidemiological, population genetics studies and for typing of the organism. Because this method is expensive to be done as a routine work in Sudan, it can be recommended for diagnosis of unusual clinical presentation as a second line confirmation in an algorithm of diagnosis.

Conclusions:

PCR is especially useful for the confirmation of cases suspected of VL. Also PCR can be performed successfully, directly from clinical samples spotted on filter papers with no prior cultivation.

Nested ITS- PCR is a more sensitive method than microscopy for the detection of leishmania parasites. In addition ITS sequences are variable and hence allow strain differences.

SSCP has the advantage over both RFLP and PCR fingerprinting that it can detect DNA polymorphisms and point mutations at a variety of positions in DNA fragments. Also it has higher resolution power for strain identification, which is required for understanding of the epidemiology of leishmania.

Anonymous regions, which are single copy genes, could be good markers especially for population genetics studies provided that we have large amount of PCR products.

Gp63 could be a potential useful marker for study of polymorphisms and the possible correlation of these polymorphisms to the severity of the disease.

Different levels of heterogeneity occur among different species of Leishmania with L. tropica being most variable (Schönian et al.; 2001) followed by L.aethiopica (Schönian et al., 2000) and then L. donovani (this study, Lewin, 2000)

It would be interesting to determine whether L. donovani causing mucosal leishmaniasis, VL and PKDL in Sudan could be correlated with specific SSCP patterns, may be other relevant genes could be used in further study using SSCP technique.